This project will study synthesis of vasoactive metabolites of arachidonic acid in cultured cells and their role in platelet aggregation. A principal objective will be to rationalize the effects of inhibitors, particularly aspirin, in order to explain anomalous clinical findings and the mechanism of unexpected side effects of aspirin. Vascular smooth muscle and endothelial cells will be used to study synthesis of the anti-platelet substance prostacyclin from 14C-arachidonic acid. Prelabelled cultures will be used to identify prostacyclin releasing-factors of physiological importance. The possibility that leukotrienes serve as releasing-factors will be examined. A specific inhibitor of the lipoxygenase, (15-HETE), synthesized in leucocytes, will be used to evaluate the role of this pathway in platelet function. Thrombin-induced prostacyclin release in vascular cells will be characterized to determine its significance to regulation of hemostasis in vivo. The molecular mechanisms underlying permanent impairment of this control system by aspirin, despite recovery of the cyclooxygenase, will be investigated. The influence of aspirin on other prostacyclin releasing-factors including calcium ionophores, melittin and histamine, will be examined. The effects of aspirin will be compared to those of reversible cyclooxygenase inhibitors including phenylbutazone, sulfinpyrazone and fenamic acids. The mechanism of stimulation of prostacyclin synthesis by dipyridamole will be studied as a possible explanation for the reputed long-term effectiveness of this drug in angina. The cell culture results will be correlated with in vivo effects by use of an aorta strip chamber technique, measuring prostacyclin release both by radioimmunoassay and by radiomass spectrometry. In addition to elucidating molecular mechanisms underlying synthesis and function of vasoactive arachidonate metabolites in the cardiovascular system, these studies will provide basic information badly needed in selecting drugs for use in the long-term therapy and prevention of cardiovascular disease.